Applications

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application

Abreviews

Notes

Dot blot

Use at an assay dependent concentration.

WB

1/1000 - 1/5000.

ELISA

1/25000. This product has been assayed against 1.0 ug of Armenian Hamster IgG in a standard capture ELISA using ABTS (2,2’-azino-bis-[3-ethylbenthiazoline-6-sulfonic acid]) as a substrate for 30 minutes at room temperature.

Electron Microscopy

Use at an assay dependent concentration.

IHC-P

1/1000 - 1/5000.

ICC

1/1000 - 1/5000.

Images

ELISA - Rabbit Anti-Armenian hamster IgG H&L (HRP) (ab5745)

ab5745 tested against purified Armenian Hamster IgG. Each well was coated in duplicate with 1.0 µg of Armenian Hamster IgG. The starting dilution of antibody was 5µg/ml and the X-axis represents the log10 of a 3-fold dilution. This titration is a 4-parameter curve fit where the IC50 is defined as the titer of the antibody. Assay performed using 3% fish gelatin as blocking buffer and TMB substrate

Ab5745 staining Armenian hamster IgG by Immunohistochemistry (IHC-P) using the spleen of a healthy C57BL/6J mouse. Tissue was fixed with formalin. Antigen retrieval was by heat mediation in citrate buffer, carried out in a pressure cooker. Tissue sections were blocked with 2.5% goat serum for 2 hours at room temperature, followed by incubation with primary antibody (ab231545 1/100 in 1% BSA) overnight at 4°C. The secondary antibody (1/300 in 1% BSA) was added for 40 min at room temperature. (The image shows the second antibody control stain for positive stain please see ab231545)

Thanks for your message and for kindly providing this further information.

I have copied below the IHC-P testing protocol for this antibody, which includes the antigen retrieval details. I hope this will be helpful. Please note this protocol will be a guideline only and may require some individual optimization.

Please do not hesitate to let me know how you get on with the next experiments. If the experiment is still not working, please do not hesitate to contact me with the following details as previously requested:

1. Please provide the order number and date of purchase.

2. Time for antigen retrieval with paraffin embedded samples will require some individual optimization depending on the individual experiment (sample type, epitope being detected, species etc). Try different time points to see which provides better results, for example 2, 5, 10 and 20 minutes.

3. TCR gamma /TCR delta is a membrane protein. Therefore permeabilization will not be required. Triton is quite a strong detergent and can disrupt membrane proteins and significantly affect the staining.

Try including a more gentle detergent, 0.2% Tween in the wash buffer and antibody dilution buffer. This will help to keep the antibody solubilised and wash away any excess antibody.

I look forward to hearing from you.

Tissue Preparation: Formalin fixation and embedding in paraffin wax

Tissue Sectioning: Make 4-um sections and place on pre-cleaned and charged microscope slides. Heat in a tissue-drying oven for 45 minutes at 60°C.

Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.

The details you have kindly provided will enable us to investigate this case for you and this is also helpful in our records for monitoring of quality.

I would like to reassure you that ab118864 is tested and covered by our 6 month guarantee for use in IHC-Fr and mouse samples. In the event that a product is not functioning in the applications cited on the product data sheet, we will be pleased to provide a credit note or free of charge replacement.

Although the other antibody has worked well using this procedure, individual antibodies will often require optimization. Protocols are provided as a guideline only and individual optimization will often be required for individual experiments. I would like to offer some suggestions to help optimize the results from ab118864. I would also appreciate if you can confirm some details of the procedure.

1. Please provide the order number and date of purchase.

2. Fixation: for frozen sections, we would recommend to fix in 4% PFA for 10 minutes only. This should mean that antigen retrieval is then not required. Could you confirm these are frozen samples? There is no mention of paraffin embedding.

3. Antigen retrieval: Antigen retrieval is not usually required for frozen sections.

Time for antigen retrieval with paraffin embedded samples will require some individual optimization depending on the individual experiment (sample type, epitope being detected, species etc). Try different time points to see which provides better results, for example 2, 5, 10 and 20 minutes.

4. TCR gamma /TCR delta is a membrane protein. Therefore permeabilization will not be required. Triton is quite a strong detergent and can disrupt membrane proteins and significantly affect the staining.

Try including a more gentle detergent, 0.2% Tween in the wash buffer and antibody dilution buffer. This will help to keep the antibody solubilised and wash away any excess antibody.

5. Please confirm what dilutions of primary antibody have been tried? I can suggest to increase the concentration to increase the signal obtained.

6. Is the secondary antibody working well with other primary antibodies? This will help to assess where the problem is.

I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details. I hope we can resolve this case for you as soon as possible.

Thank you for your enquiry.
This product ab36772 was created by immunizing Armenian hamsters with a synthetic peptide conjugated to KLH. The resulting splenocytes were then fused to the tumor cell line SP2/0.
We recommend using any HRP conjugated, anti-hamster Ig secondary to detect this product. by Western blot. Also, a secondary antibody that is specifically against Armenian hamster, like ab5745 should be a very good secondary too.
If there is anything else that I can help you with, please do not hesitate to contact me.